RESEARC H Open Access TTF-1 regulates a 5 nicotinic acetylcholine receptor (nAChR) subunits in proximal and distal lung epithelium Paul R Reynolds * , Camille H Allison , Charles P Willnauer Abstract Background: Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels comprised of five similar subunits that influence signal transduction and cell turnover. a 5 is a structural subunit detected in many non- neuronal tissues; however, its function during pulmonary development is unknown. Results: a 5 was assessed by immunohistochemistry and RT-PCR in mouse lungs from embryonic day (E)13.5 to post-natal day (PN)20. From E13.5 to E18.5, a 5 expression was primarily observed in primitive airway epithelial cells while mesenchymal expression was faint and sporadic. a 5 expression was detected throughout the proximal lung at PN1 and extensively expressed in the peripheral lung at PN4, an early stage of murine alveologenesis. An interesting shift occurred wherein a 5 expression was almost undetectable in the proximal lung from PN4-PN10, but significant localization was again observed at PN20. Transcriptional control of a 5 was determined by assessing the activity of reporters containing 2.0-kb and 850-bp of the mouse a 5 promoter. Because perinatal expr ession of a 5 was ab undant in bronchiolar and alveolar epithelium, we assessed transcriptional control of a 5 in Beas2B cells, a human bronchiolar epithelial cell line, and A-549 cells, an alveolar type II cell-like human epithelial cell line. Thyroid Transcription Factor-1 (TTF-1), a key transcription regulator of pulmon ary morphogenesis, significantly increased a 5 transcription by acting on both the 2.0-kb and 850-bp a 5 promoters. Site-directed mutagenesis revealed that TTF-1 activated a 5 transcription by binding specific TTF-1 response elements. Exogenous TTF-1 also significantly induced a 5 transcription. Conclusions: These data demonstrate that a 5 is specifically controlled in a temporal and spatial manner during pulmonary morphogenesis. Ongoing research may demon strate that precise regulation of a 5 is important during normal organogenesis and misexpression correlates with tobacco related lung disease. Background Mechanisms that control pulmonary development involve highly coordinated processes that require precise reciprocal interactions between endodermally derived respiratory epithelium and the surrounding splanchnic mesenchyme. These interactions are predominantly mediated by cell surface receptors and specific ligands elaborated by communicating cells of both germinal ori- gins. Initial primordial lung buds undergo branching to form the main bronchi and extensive subsequent branching e vents lead to the formation of the intrapul- monary conducting and peripheral lung airways. Distinct populations of differentiated respiratory epithelial cell types then arise, producing a morphologically dynamic arrangement of cells that in due course i nfluence pul- monary function and respiratory efficiency. The tem- poral and spatial pattern of cell surface receptor expression must therefore be specifically controlled in order to orchestrate mechanisms of proliferation, migration, and differentiation essential during lung morphogenesis. Thyroid transcription factor (TTF)-1 is a member of the hom eodomain-containing Nkx2 family of transcrip- tion factors. TTF-1 is expressed in the lung, thyroid, ventral forebrain, and the pituitary [1-3]. While TTF-1 mRNA is initially detected in the mouse at E10 [ 4] its pattern of expression principally localizes to the lung * Correspondence: paul_reynolds@byu.edu Department of Physiology and Developmental Biology, Brigham Young University, Provo, UT 84602, USA Reynolds et al. Respiratory Research 2010, 11:175 http://respiratory-research.com/content/11/1/175 © 2010 Reynolds et al; licensee BioMed Central Ltd. This is an Open Access art icle distributed under the terms of the Creative Commons Attribution License (ht tp://creative commons.org/licenses/by/2.0), which permits unrestricted use, distributio n, and reproduction in any medium, provided the original work is properly cited. periphery during pulmonary development [2]. TTF-1 activates the expression of genes critical to lung devel- opment and function such as surfactant proteins (SPs), Clara cell secretory protein (CCSP), various growth fac- tors, and molecules required for normal host defense and vasculogenesis [4,5]. Inactivation of TTF-1 causes tracheo-esophageal fistulae and impairment of pulmon- ary branching, leading to severe lung hypoplasia [6]. In concert with other transcription factors, TTF-1 binds TTF-1 response elements (TREs) i n promoters of target genes i n order to regulate gene expression and cell dif- ferentiation during lung morphogenesis. Whil e our pre- liminary studies and the work of others reveal that a 5 is detected in cells known to express TTF-1 [7-9 ], no reg- ulatory mechanism has been proposed linking the two in the lung to date. Neuronal and non-neuronal nicotinic acetylcholine receptors (nAChRs) combine with glycine, GABA A ,and 5HT3 receptors to form a family of ligand-gated ion channels [10]. nAChRs are pentameric oligomers com- posed of f ive related subunits arranged around a central ion channel that allows flow of calcium or sodium fol- lowing ligand binding. Subsequent to ligand interaction, pathways associated with intracellular signal transduc- tion, proliferation, and apoptosis are induced [11-13]. Several receptor subunits have been identified and are classified as either agonist binding (a 2 , a 3 , a 4 , a 6 , a 7 , a 9 and a 10 ) or structural (a 5 , b 2 , b 3 and b 4 ) [14,15]. Work performed previously by our laboratory demonst rated that a 7 nAChRs, homomeric receptors composed of five a 7 subunits, are temporally controlled in the lung dur- ing development and are transcriptionally regulated by TTF-1 [16]. In the current investigation, we report that a 5 nAChR subunits are expressed in subsets of pulmonary epithe- lial c ells during stages of lung morphogenesis and that these receptor subunits are regulated by TTF-1. T his research adds additional insight into TTF-1 regulation of subunits involved in nAChR assembly by joining a 5 and a 7 in conserved regulatory pathways. Furthermore, because comparisons between the human a 5 gene and the a 5 gene in several other species reveal remarkable conservation, TTF-1 and its homologues may be com- mon transcriptional regulators involved i n controlling the precision of a 5 nAChR expression in the lung. Methods Mouse Models a 5 expression was assessed from E13.5 to PN20 in lungs from wild type and TTF-1 null mice, each in a C57Bl/6 bac kground. Dr. Jeffrey Whitsett at the Cincinnati Chil- dren’s Hospital Medical Center (CCHMC) generously gifted TTF-1 null mice. Animal husbandry and use fol- lowed protocols approved by the Institutional Animal Care and Use Committee at CCHMC and Brigham Young University. Antibodies A rabbit a 5 polyclonal antibody (generated and kindly gifted by Scott Rogers and Lorise Gahring at the Univer- sity of Utah) was generated against epitopes in the cyto- plasmic domain of the a 5 protein and has been demonstrated to interact with tissues emb edded in par- affin [17,18]. Antibody specificity was conf irmed using immunoblotting and ELISA, revealing that the anti- serum reacts only with the a 5 subunit protein to which it was made [19]. While data revealing positive immu- nostaining for a subset of nAChR subunits in brain sam- ples from both wild type and subunit null animals exists [20,21], there are no published reports demonstrating such effects in lung tissue or employing a 5 specific anti- bodies. The a 5 IgG was used at a dilution of 1:800. A rabbit polyclonal antibody raised against Clara Cell Secretory Protein (CCSP) generated at the CCHMC was used at a dilution of 1:1600 to identify Clara cells in the conducting airways. A rabbit polyclonal antibody for TTF-1 was also generated at CCHMC and used to loca- lize type II alveolar epithelial cells (ATII) at a dilution of 1:1000. Specificity of the CCSP and TTF-1 antibodies was determined by Western blot analysis (not shown). Immunohistochemistry Immunohistochemica l staining for a 5 , CCSP, and TTF-1 were performed using standard techniques [22,23]. Briefly, 5-μm paraffin section s from six mice per group were deparaffinized and rehydrated. Sections were trea- ted with 3% hydrogen peroxide in methanol for 15 min to quench endogenous peroxidase. Development in NiDAB was followed by incubation in Tris-cobalt, which enhanced antigen localization, and by counterstaining with nuclear fast red. Sections were then dehydrated in a series of ethanols, washed in three changes of xylene, and m ounted under coverslips with Permo unt. Control sections were incubated in blocking serum alone. Plasmid Construction and Mutagenesis Primers were designed to retrieve 2.0-kb or 0.85-kp of the mouse a 5 promoter by polymerase chain reaction (PCR) using the Expand High F idelity PCR System (Roche, Indianapolis, IN). The amplified a 5 promoter fragment was directional ly cloned into the pGL4.10- basicluciferasereporterplasmid (Promega, Madison, WI) and verified by sequencing. Site-directed mutagen- esis of potential TTF-1 binding sites was performed by using the reporter construct ( pGL4.10-0.85-kb a 5 ) and the QuickChange™ Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). Briefly, synthetic oligonucleo- tides containing the desired mutation f or TTF-1 Reynolds et al. Respiratory Research 2010, 11:175 http://respiratory-research.com/content/11/1/175 Page 2 of 10 (CAAT®GGGG) were extended during PCR amplifica- tion. The products were digested with DpnItoremove the wild-type DNA. The nicked vector DNA was then transformed into XL1-blue supercompetent cells and repaired. All constructs were verified by nucleotide sequencing. Transfection and Reporter Gene Assays Functional ass ays of reporter gene constructs were per- formed by transient transfection of Beas2B and A-549 cells u sing FuGENE-6 reagent (Ro che). Beas2B is a transformed human bronchiolar epithelial cell line and A-549 i s a human pulmonary adenocarcinoma cell line characteristic of ATII cells. Cells in 35- mm dishes at 40-50% confluence were transfected with four plasmids at the following concentrations: 300 ng pRSV-bgal, 100 ng pGL4.10-2.0-kb a 5 or pGL4.10-0.85-kb a 5 , 100-400 ng pCMV-TTF-1 and pCDNA control vector to bring total DNA concentration to 1.2 μg. The cells were allowed to grow to confluence (48 hr), washed with cold PBS, lysed, and snap frozen for several hours. The plates were scraped and centrifuged, and the cleared superna- tant was u sed for both b -gal and luciferase assays. Reporter assays were normalized for transfection effi- ciency based on the b-gal activity [22]. Luciferase activ- ity was determined in 10 μl of extract at room temperature with 100 μl of luciferase substrates A and B (BD Biosciences, San Jose, CA) for 10 sec after a 2-sec delay in a Moonlight™ 3010 luminometer (BD Biosciences). RT-PCR In order to assess a 5 mRNA expression throughout developm ent, total RNA was isola ted from whole mou se lungs at various time points with the Absolutely RNA® RT-PCR Miniprep Kit (Stratagene) and DNase treated. Because a 5 was immunolocalized in bronchioles and alveoli, induction of a 5 mRNA expression was similarly assessed in Beas2B and A-549 cells following transfec- tion with 400 ng pCMV-TTF-1 or control pCDNA vec- tor. 2-μg of total RNA was reverse transcribed using the SuperScript® III First- Strand Kit according to the manu- facturer’s instructions (Invitrogen). PCR was performed with 2-μ laliquotsofthegeneratedcDNAusingTaq polymerase (Roche, Indianap olis, IN) and experiments included no template (lacking cDNA) and no RT (with- out reverse transcriptase) controls (not shown). Products were electrophoresed on a 1.5% agarose gel with appro- priate molecular weight standards. Bands were quanti- fied using Un-Scan-it™ gel digitizing software (Silk Scientific, Orem, UT). Gene expression was assessed in three replicate p ools and representative data is shown. Primers u sed for the P CR reactions include a 5 forward (5’-CTT CAC ACG CTT CCC AAA CT-3’) and reverse (5’ -CT T CAA CAA CCT CAC GGA CA-3’ )and GAPDH forward (5’ -CGT CTT CAC CAC CAT GGA GA-3’) and reverse (5’-CGG CCA TCA CGC CAC AGC TT-3’). PCR parameters included an initial heating at 94°C for 5 m. a 5 and GAPDH were ampl ified vi a 30 cycles at 94°C for 45 s, 57°C for 45 s, and 72°C for 45 s. All amplifications were followed by a 7-min exten- sion at 72°C. Statistical Analysis Results are presented as the means ± S.D. of six repli- cate pools per group. Means were assessed by one and two-way analysis of variance (ANOVA). When ANOVA indicated significant differences, student t tests were used with Bonferroni correction for multiple compari- sons. Results are representative and those with p values < 0.05 were considered significant. Results a 5 nAChR Expression During Lung Development The distribution of a 5 was assessed in mouse lung by immunohistochemistry from E13.5 to PN20. At E13.5 (Figure 1A) and E15.5 (Figure 1B), a 5 was predomi- nantly observed in primitive airway epithelial cells and sporadically detected in pulmonary mesenchyme. While mesenchymal staining diminished through E18.5, a 5 expression in pulmonary epithelium increased and was restricted to luminal cell surfaces (Figure 1D). a 5 expression was abundantly detected in proximal lung epithelial cells at PN1 (Figure 1E), however, by PN4, a 5 expression was only detected in the peripheral respira- tory region of the lung (Figure 1F). Staining at PN4, a period that coincides with the onset of alveologenesis, revealed a 5 expression in cells located near alveolar septa characteristic of ATII localization. This pattern of expression in respiratory epithelial cells and minimal to no staining in the proximal lung persisted through PN10 (Figure 1G). BY PN20, a 5 expression was detected throughout the lung, with abundant immunolocalization in proximal airway epitheliumaswellasintherespira- tory compartment (Figure 1H). No staining was observed in sections stained without primary antibody (Figure 1I). The patterns of a 5 expression obtained by immunostaining corresponded with a 5 mRNA expres- sion from E13.5 to PN20 as revealed by semi-quantita- tive RT-PCR analysis (Figure 2). To identify epithelial cells that express a 5 , immuno- histochemistry was performed on serial sect ions at PN1. Staining serial sections with TTF-1 (Figure 3A), an ATII cell marker, and a 5 (Figure 3B), revealed a 5 expression in ATII cells w ith nuclear staining for TTF-1. While a 5 was expressed in many ATII cells (Figure 3A and 3B, arrows) not all ATII cells were identified with a 5 stain- ing. Localization in serial sections was also performed Reynolds et al. Respiratory Research 2010, 11:175 http://respiratory-research.com/content/11/1/175 Page 3 of 10 with CCSP, a Clara cell specific marker that identifies non-ciliated Clara cells in the proximal lung that slightly protrude into the airway lumen (Figure 3C). a 5 staining (Figure 3D) appeared to be associated with many CCSP- secreting Clara cells in pulmonary bronchioles (Figure 3C). TTF-1 Regulates a 5 Transcription In Vitro Because there were interesting shifts in the expression of a 5 by ATII cells at various developmental time points (Figure 1E,F,G,H), experiments were p lanned that tested whether TTF-1 transcriptionally regulates a 5 expression. An assessment of the mouse a 5 promoter sequence revealed the locations of nine potential TTF-1 regulatory elements (TREs) in the 2.0-kb promoter fragment and five in the 0.85-kb fragment (Figure 4A). Because a 5 experienced profound expression changes from proximal lung (Figure 1E) to distal lung (Figure 1F and 1G) before returning to the proximal lung (Figure 1H), we tested the degree of TTF-1 regulation in both bronchiolar epithelium (Beas2B) and ATII-like alveolar epithelial cells (A-549). TTF-1 (100-400 ng) activated the 2 .0-kb a 5 promoter in a dose-dependent manner in both Beas2B and A549 cells (Figur e 4B and 4C). TT F-1 also significantly induced transcription of a 5 in both cell types when a truncated reporter that contains only Figure 1 Immunostaining revealed distinct patterns of a 5 expression in the lung during organogenesis.A.a 5 was expressed at E13.5 in primitive airway epithelium (arrow) and sporadically in pulmonary mesenchyme (arrowhead). B and C. During the late pseudoglandular period of development (B, E15.5) and early saccular stage of development (C, E17.5), a 5 continued to localize to pulmonary epithelium (arrows). D. At E18.5, a 5 expression was detected in larger airways (arrows) as well as in primitive respiratory epithelium (arrowhead). E. At PN1, a 5 expression was markedly detected in proximal lung airways (arrow) and only minimally detected in the peripheral lung (arrowhead). F. At the commencement of alveologenesis (PN4), a 5 localized to lung parenchyma (arrow) and was noticeably absent in the airways (arrowhead). G. At PN10, a 5 expression persisted in the respiratory compartment (arrow). H. After alveologenesis progressed to PN20, a 5 was abundantly expressed throughout the lung, being detected in proximal (arrow) as well as distal pulmonary epithelium (arrowhead). I. PN20 sections incubated without primary a 5 antibody revealed no immunoreactivity. Six mice were included in each group and representative images at 40× magnification are shown. Reynolds et al. Respiratory Research 2010, 11:175 http://respiratory-research.com/content/11/1/175 Page 4 of 10 0.85-kb of the promoter was transfecte d (Figure 4D and 4E). To identify potential TREs that are critical in TTF-1- mediated control of a 5 transcription, site-directed muta- tional analysis was performed resulting in the ablation of putative TREs (Figure 5A). In Beas2B cells, mutation of the second and fourth TRE resulted in a significant decrease in TTF-1-induced transcription compare d to the wild type promo ter transfected with TTF-1 ( Figure 5B). Experiments involving A-549 cells re vealed that mutation of any of the five TREs resulted in a significant decrease in TTF-1-induced transcription (Figure 5B). TTF-1 Mediates a 5 Expression In Vitro In order to further assess the effects of TTF-1 on a 5 expression, Beas2B a nd A-549 cells were transfected with TTF-1 and a 5 was assessed by RT-PCR. In the absence of exogenous TTF-1, Beas2B and A-549 cells bot h expressed detectible levels of a 5 (Figure 6). Trans- fection of TTF-1 24 hours before RNA isolation induced a significant increase in a 5 mRNA expression in both A-549 and Beas2B cells (Figure 6). TTF-1 Targeting Impairs a 5 Expression In Vivo Because our data demonstrate that a 5 is expressed in pulmonary epithelium at E18.5 (Fi gure 1D) and its expression is regulat ed by TTF-1 (Figures 4, 5, 6), we determined a 5 expression in TTF-1 null mice. TTF-1 null mice die at birth due to significantly reduced branching morphogenesis and severe l ung hypoplasia. Expression of a 5 in pulmo nary epithelium in the lungs of TTF-1 null mice (Figure 7A, arrow) was nearly unde- tectable when compared to intense a 5 localization observed in age-matched wild type control lung samples (Figure 7B, arrows). Discussion and Conc lusions The temporal-spatial distribution of a 5, a member of the nicotinic acetylcholine receptor subunit family, was determined during embryonic and postnatal lung devel- opment. Various epithelial cell pop ulations expressed a 5 protein in both the conducting and peripheral air spaces. a 5 was primarily expressed in respiratory epithe- lial cells during the embryonic, pseudoglandular, canna- licular, and saccular stages of lung development. In addition to expression in the peripheral lung, a 5 was also detected perinatally in distinct populations of bronchiolar epithelial cells. Conducting airway epithelial cell expression persisted throughout lung morphogenesis except from PN4 to PN10, a period that coincides with significant parenchymal differentiation in the alveolar stage of lung development. Immunolabeling of a 5 in the fetal lung was observed primarily on lumin al epithelial cell membranes suggesting that a 5 accumulates on api- cal cell surfaces in order assemble receptors needed in the postnatal lung. Alternatively, apical expression may suggest that a 5 subunits combine in utero to form func- tional nAChRs which bind ligand and signal event s that are esse ntial during organogenesis. Several groups have shown t hat nAChRs are expressed in airway epithelium and that they form functional receptors as demonstrated by electrophysiological analyses [8,24,25]. Localization of a 5 with cells that express CCSP and TTF-1 suggests that a 5 is regulated by TTF-1 and, therefore, may play a role in the mediation of paracrine signaling between respiratory epithelial cells during pulmonary morphogenesis. Intriguing aspects of functional pulmonary nAChRs in utero are data related to acetylcholine (ACh) as a local signaling molecule synthesized by many non- neuronal cells [26]. In order for ACh to function as a signal in the lung, ACh must be synthesized and secreted locally. Choline is incorporated into pul monary bronchiolar cells by a choline high-affi nity transporter (CHT), synthesized into ACh by choline acetyl transfer- ase (ChAT), and packaged into transport vesicles by a vesicular ACh transporter (VAChT) [26]. Availability of choline in the lung is also possible due to its derivation during the recycling of surfacta nt proteins and mem- branes [27]. In addition to acetylation during the gen- eration of ACh, choline has also been demonstrated to be an agonist for a subset of ligand binding nAChR sub- units such as a 7 [28]. While evidence for choline and acetylcholine ligation primarily identifies with the Figure 2 a 5 mRNA expression in the lung during organogenesis. Semi-quantitative RT-PCR analysis revealed variable abundance of a 5 mRNA expression in whole mouse lung from E13.5 to PN20. Band densities were assessed and normalized after standardization of GAPDH band densities to 1. Although samples were screened from 6 mice at each time point, only a representative single band is shown. Reynolds et al. Respiratory Research 2010, 11:175 http://respiratory-research.com/content/11/1/175 Page 5 of 10 Figure 3 a 5 was expressed by and ATII cells Clara cells at PN1. Staining for TTF-1 (A) and a 5 (B) in serial sections from PN1 mouse lung revealed a 5 is co-expressed with TTF-1 in many ATII cells, but not all (arrows). Staining for CCSP (C) and a 5 (D) also identified consistent a 5 expression in bronchiolar epithelium at PN1 (arrows). All images are at 80× original magnification. Figure 4 TT F-1 activated a 5 transcription in bronchiolar and alveolar epithelial cell types.A.Schematicofa 5 luciferase reporters containing 2.0-kb or 0.85-kb mouse a 5 promoter sequences that include putative TTF-1 response elements (TREs, black rectangles). B and C. TTF- 1 dose-dependently induced a 5 transcription by acting on a 2.0-kb a 5 reporter in Beas2B (B) and A-549 (C) cells. D and E. TTF-1 also induced significant increases in a 5 transcription via interaction with a truncated 0.85-kb a 5 reporter. Significant differences in luciferase levels compared to reporter alone are noted at P ≤ 0.05 (*). Reynolds et al. Respiratory Research 2010, 11:175 http://respiratory-research.com/content/11/1/175 Page 6 of 10 biology of a 7 , the possibility exists that similar agonists interact with receptors structurally maintained by a 5 . a 5 was co-expressed with TTF-1 in epithelial cells that contribute to primordial tubules early in lung develop- ment [29]. TTF-1 regulates cytodifferentiation and for- mation of functional respiratory epithelium [5]. Several additional co-expressed transcriptional regulators such as GATA-6 and FoxA2 are also observed in airway epithelium during the period from E13.5 to 15.5 [3,30]. Recent preliminary studies performed in our laboratory reveal that GATA-6 and FoxA2, both transcriptional targets of TTF-1, also individually and synergistically activate the a 5 promoter, suggesting complex interplay between TTF-1 and other important transcription fac- tors. TTF-1 and various co-regulators such as GATA-6 and FoxA2 interact during the regulation of specific genes critical to lung function, including CCSP, surfac- tant proteins, growth factors, and VEGFa and VEGFr2 essential in vasculogenesis [31]. While additional research is still necessary, the observations that a 5 was transcriptionally induced by TTF-1 via interaction with specific promoter response elements and significantly diminished in TTF-1 null mouse lung reveals the impor- tance of TTF-1 in the orchestration of a 5 regulation. This research also demonstrates that a 5 and other nAChR subunits such as a 7 [16] may contribute to an expanding group of important developmental genes regulated by TTF-1. Furthermore, because the a 5 gene and message maintain remarkable conservation across species (Table 1), TTF-1 and its homologues may Figure 5 TTF-1 induced a 5 transcription via interaction with putative TTF-1 response elements (TREs). A. Schematic of a wild type (WT) a 5 luciferase reporter containing the 0.85-kb mouse a 5 promoter sequence and reporters that contain a 0.85-kb a 5 promoter with each sequential TRE targeted by site-directed mutagenesis (Mutants A-E). B. TTF-1-mediated increases in a 5 transcription were significantly diminished in Beas2B cells when the second or fourth TREs were mutated. C. Each TRE was demonstrated to be significant in regulating TTF-1-mediated a 5 transcription in ATII-like A-549 cells. Significant decreases in TTF-1 induced luciferase activity resulting from each mutant reporter compared to WT + TTF-1 are noted at P ≤ 0.05 (*). Reynolds et al. Respiratory Research 2010, 11:175 http://respiratory-research.com/content/11/1/175 Page 7 of 10 Figure 6 a 5 mRNA express ion was induced by TTF-1 in ATII-like A-549 cells and bronchiolar epithelium-like Beas2B cells.A.Bysemi- quantitative RT-PCR analysis, A-549 and Beas2B cells endogenously express a 5 . Transfection with a TTF-1 expression vector 24 hours prior to mRNA isolation, reverse transcription, and PCR amplification resulted in detectible increases in a 5 expression. Representative examples are shown. B. Band densities from six replicates per group were assessed and normalized after standardizing GAPDH band density to 1. When all six replicates were assessed, a significant difference in a 5 expression was detected between TTF-1 and mock transfected cells (*P ≤ 0.05). Figure 7 a 5 expression was significantly reduced in pulmonary epithelium from E18.5 TTF-1 null mice compared to age-matched with type controls.A.a 5 immunostaining in TTF-null mouse lung revealed almost complete ablation of a 5 expression in pulmonary epithelium (arrow). B. Staining for a 5 demonstrated marked expression in proximal and distal pulmonary epithelium (arrows). All images are at 40× original magnification. Reynolds et al. Respiratory Research 2010, 11:175 http://respiratory-research.com/content/11/1/175 Page 8 of 10 influence common transcriptional mechanisms involved in the defined temporal and spatial pattern of a 5 nAChR expression in the lung. Even though TTF-1 specifically induced significant a 5 expression in pulmonary epithelium, the t emporal- spatial distribution of TTF-1 and a 5 during lung develop- ment were not completely identical. For example, whereas TTF-1 is an epithelium-specif ic transcription factor, a 5 protein was expressed in both the epithelium and mesenchyme at E13.5. The expression of a 5 is therefore likely regulated by the activity of several transcription fac- tors with overlapping expression patterns. Because TTF-1 regulates target gene expression in concert with other reg- ulatory factors including GATA-6, FoxA2, NF-1, RAR, and AP-1 [31], it is likely that the temporal-spatial distri- bution of a 5 expression is influenced in a complex manner by a host of transcription factors. Nicotinic cholinergic signaling via a 5 nAChR subunits in airway epithelial cells is likely affected by nicotine. Published reports demonstrate that cells exposed to environmental tobacco smoke, or equal concentrations of nicotine, induce sequential severalfold increases in a 5 and a 7 expression [32]. Plasma nicotine levels in smo- kers fluctuate between 10 and 200 nM and epithelial cells directly exposed to smoke may experience nicotine levels that are 5-10-fold greater [33,34]. Exposure to cigarette smoke duri ng pregnancy adversely affects lung development as manifested by significantly reduced branching morphogenesis [35], increased respiratory ill- ness [36], altered pulmonary function [37], and perma- nent airway obstruction in the proximal lung [38]. Nicotine crosses the placenta and directly affects lung development in ut ero via interaction with nAChRs in the developing and post-nata l lung. Our studies demon- strate that while receptors that contain a 5 are expressed in populations of epithelial cells during lung develop- ment, receptor availability may contribute to adverse lung development and morphological perturbation when noxious ligands are present. Recently the a 5 gene (CHRNA5) and other receptor subunits located in the chromosome 15q24-25 region have been the topics of intense investigation due to a correlation between an a 5 variant and nicotine depen- dence [39]. While research is ongoing, analysis of this specific chromosomal locus reveals that a 5 and its var- iants significantly influence susceptibility to smoke related lung cancer and chronic obstructive pulmonary disease (COPD) [39-41]. Understanding the develop- mental role of a 5 and TTF-1-mediated mechanisms that control its precise pattern of expression during lung organogenesis will prove beneficial in elucidating the role of a 5 in the progression of lung disease commenced in utero by tobacco exposure. In conclusion, a 5 nAChR subunits are expresse d in specific epithelial cell types in the lung during develop- ment. a 5 expression is developmentally regulated by sev- eral factors including TTF-1, a molecule centrally involved in normal lung formation. Our data reveals specific regulation of a 5 expression by TTF-1; however, such expression may be altered by nicotine exposure. While nicotine may dire ctly influence normal choliner- gic signaling during morphogenesis that involves a 5 - containing nAChRs, the misregulation of a 5 may also predispose individuals to lung cancer and COPD. Acknowledgements The authors wish to thank Dr. Jeffrey Whitsett from the Cincinnati Children’s Hospital Medical Center (CCHMC) for providing lung samples from TTF-1 null mice. Scott W. Rogers and Lorise C. Gahring (University of Utah) kindly provided the a 5 rabbit polyclonal antibody. This work was supported by the Flight Attendant’s Medical Research Institute (FAMRI, PRR) and a BYU Mentoring Environment Grant Award (PRR). Authors’ contributions CHA generated plasmids and assisted with in vitro reporter gene assays. CPW performed immunohistochemistry, reporter gene assays, RT-PCR analysis and assisted in manuscript preparation. PRR conceiv ed of the study and supervised in its implementation, interpretation, and writing. All authors approved of the final manuscript. Competing interests The authors declare that they have no competing interests. Received: 29 July 2010 Accepted: 9 December 2010 Published: 9 December 2010 References 1. Guazzi S, Proce M, DeFelice M, Damante G, Mattei MG, DiLauro R: Thyroid nuclear factor 1 (TTF-1) contains a homeodomain and displays a novel DNA binding specificity. EMBO J 1990, 9:3631-3639. 2. Lazzaro D, Proce M, DeFelice M, DeLauro R: The transcription factor TTF-1 is expressed at the onset of thyroid and lung morphogenesis and in restricted regions of the foetal brain. Development 1991, 113:1093-110. 3. Mizuno K, Gonzales FJ, Kimura S: Thyroid-specific enhancer-binding protein (T/EBP): cDNA cloning, functional characterization, and structural identity with thyroid transcription factor TTF-1. Mol Cell Biol 1991, 11:4927-33. 4. 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Bierut LJ: Convergence of genetic findings for nicotine dependence and smoking related diseases with chromosome 15q24-25. Trends Pharmacol Sci 2009, 31:46-51. 40. Spitz MR, Amos CI, Dong Q, Lin J, Wu X: The CHRNA5-A3 region on chromosome 15q24-25.1 is a risk factor both for nicotine dependence and for lung cancer. J Natl Cancer Inst 2008, 100:1552-1556. 41. Pillai SG, Ge D, Zhu G, Kong X, Shianna K, Need A, Feng S, Hersh C, Bakke P, Gulsvik A, Ruppert A, Lodrup C, Roses A, Anderson W, Investigators ICGN, Rennard SI, Lomas DA, Silverman EK, Goldstein DB: A genome-wide association study in chronic obstructive pulmonary disease (COPD): identification of two major susceptibility loci. PLoS Genet 2009, 5:e1000421. doi:10.1186/1465-9921-11-175 Cite this article as: Reynolds et al.: TTF-1 regulates a 5 nicotinic acetylcholine receptor (nAChR) subunits in proximal and distal lung epithelium. Respiratory Research 2010 11:175. Reynolds et al. Respiratory Research 2010, 11:175 http://respiratory-research.com/content/11/1/175 Page 10 of 10 . Access TTF-1 regulates a 5 nicotinic acetylcholine receptor (nAChR) subunits in proximal and distal lung epithelium Paul R Reynolds * , Camille H Allison , Charles P Willnauer Abstract Background: Nicotinic. eodomain-containing Nkx2 family of transcrip- tion factors. TTF-1 is expressed in the lung, thyroid, ventral forebrain, and the pituitary [1-3]. While TTF-1 mRNA is initially detected in the. additional insight into TTF-1 regulation of subunits involved in nAChR assembly by joining a 5 and a 7 in conserved regulatory pathways. Furthermore, because comparisons between the human a 5 gene and the